Identification and Quantification of 5-Methylcytosine and 5-Hydroxymethylcytosine on Random DNA Sequences by a Nanoconfined Electrochemiluminescence Platform
Mao-Hua Gao, Mei-Chen Pan, Pu Zhang, Wen-Bin Liang, Xia Zhong*, and Ying Zhuo*
Cite this: Anal. Chem. 2023, 95, 25, 9598–9604
Publication Date:June 13, 2023
https://doi.org/10.1021/acs.analchem.3c01252


Abstract


5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are two of the most abundant epigenetic marks in mammalian genomes, and it has been proven that these dual epigenetic marks give a more accurate prediction of recurrence and survival in cancer than the individual mark. However, due to the similar structure and low expression of 5mC and 5hmC, it is challenging to distinguish and quantify the two methylation modifications. Herein, we employed the ten-eleven translocation family dioxygenases (TET) to convert 5mC to 5hmC via a specific labeling process, which realized the identification of the two marks based on a nanoconfined electrochemiluminescence (ECL) platform combined with the amplification strategy of a recombinase polymerase amplification (RPA)-assisted CRISPR/Cas13a system. Benefiting from the TET-mediated conversion strategy, a highly consistent labeling pathway was developed for identifying dual epigenetic marks on random sequence, which reduced the system error effectively. The ECL platform was established via preparing a carbonized polymer dot embedded SiO2 nanonetwork (CPDs@SiO2), which exhibited higher ECL efficiencies and more stable ECL performance compared to those of the scattered emitters due to the nanoconfinement-enhanced ECL effect. The proposed bioanalysis strategy could be employed for the identification and quantification of 5mC and 5hmC in the range from 100 aM to 100 pM, respectively, which provides a promising tool for early diagnosis of diseases associated with abnormal methylation.

5-甲基胞嘧啶（5mC）和5-羟甲基胞嘧啶是哺乳动物基因组中最丰富的两种表观遗传标记，并且已经证明，这些双重表观遗传标志比个体标记更准确地预测癌症的复发和存活。然而，由于5mC和5hmC的结构相似且低表达，区分和量化这两种甲基化修饰具有挑战性。在此，我们使用了十个十一位易位家族的双加氧酶（TET），通过特定的标记过程将5mC转化为5hmC，这实现了基于纳米限制电化学发光（ECL）平台的两个标记的识别，并结合了重组酶聚合酶扩增（RPA）辅助的CRISPR/Cas13a系统的扩增策略。得益于TET介导的转化策略，开发了一种高度一致的标记途径来识别随机序列上的双重表观遗传标记，有效地减少了系统误差。ECL平台是通过制备碳化聚合物点嵌入SiO2纳米网络而建立的(CPDs@SiO2)，由于纳米约束增强的ECL效应，与散射发射体相比，其表现出更高的ECL效率和更稳定的ECL性能。所提出的生物分析策略可用于分别鉴定和定量100aM至100pM范围内的5mC和5hmC，这为早期诊断与异常甲基化相关的疾病提供了一个有前途的工具。

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